【作者】 张凯；

【导师】 张运；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 1研究背景动脉粥样硬化(Atherosclerosis, AS)不仅可以累及体循环的大中动脉也可累及前小动脉和小动脉导致微血管病变,这些血管病变可引起多个器官的形态和功能异常,其中,心、脑、肾是主要的AS靶器官,可导致心肌梗死、缺血性心肌病,脑卒中和慢性肾病,最终导致多器官功能衰竭和死亡。因此,深入研究AS大血管和微血管病变的发生机制和干预靶点,对于AS的防治具有重要的理论和实际意义。九十年代以来大量研究显示,AS易损斑块的形成是急性心脑血管事件的主要原因,因此,斑块易损性也成为国内外研究的热点,其主要的病理学特点包括较薄的纤维帽,较大的脂质核心和大量炎性细胞浸润。由于胶原是纤维帽的主要构成成分,因此斑块内胶原代谢紊乱是影响斑块易损性的重要因素。4-羟基-脯氨酸羟化酶(P4H)是所有已知类型的胶原合成过程中的关键酶,能够催化位于X-脯氨酸-甘氨酸(X-Pro-Gly)重复序列中的脯氨酸变为羟脯氨酸,该反应在前胶原多肽链折叠成稳定的三螺旋结构的过程中发挥重要作用。P4H的同工酶P4Ha1在多种组织中均有表达,其表达减少可导致斑块内胶原含量减少,进而导致AS斑块不稳定,因而是决定斑块稳定性的重要因素。既往研究发现多种因素均能影响P4Ha1的表达,例如TGFβ1能够上调P4Ha1的表达而吸烟可抑制其表达；本实验室也曾发现炎症因子TNF-α和IL-6分别通过ASK1-JNK-NonO和ERK1/2-Sp1信号通路抑制P4Ha1的表达。氧化型低密度脂蛋白(oxidized low density lipoprotein, ox-LDL)是动脉粥样硬化形成和发展过程中的重要危险因素。研究发现ox-LDL能够激活基质金属蛋白酶(matrix metalloproteinases, MMPs),增加胶原降解,进而诱导斑块不稳定。但是,ox-LDL能否通过调节P4Hα1的表达进而调节胶原合成目前尚未见报道。他汀类药物不仅具有降脂作用,还具有多种有益于心血管疾病的作用。大量研究证实他汀类药物能够增加斑块内胶原含量进而增加斑块稳定性,其机制为他汀类药物抑制MMPs的表达和活性以及增加了基质金属蛋白酶组织抑制因子-1(tissue inhibitors of metalloproteinase-1,TIMP-1)的表达。然而,他汀类药物能否通过调节P4Hα1的表达进而调节胶原合成目前尚未见报道。2研究目的(1)探讨ox-LDL对P4Hα1的抑制作用及其机制并探讨辛伐他汀的干预作用；(2)观察辛伐他汀干预对斑块内P4Hα1表达的影响及并探讨其机制,揭示他汀类药物稳定动脉粥样硬化斑块的新靶点。3实验方法3.1细胞培养与处理(1)在时间-效应实验中,我们用50ug/ml的ox-LDL刺激平滑肌细胞0、4、8、12和24小时,为保证ox-LDL不至于因为刺激时间过长而降解或者失活,每隔8小时更换含有50μg/ml的ox-LDL的新鲜培养基；在剂量-效应实验中,0、25、50ug/ml的ox-LDL分别刺激平滑肌细胞8小时,收集细胞。(2)50ug/ml的ox-LDL或者50ug/ml的ox-LDL+辛伐他汀分别刺激细胞0分钟、5分钟、15分钟、30分钟、2小时、4小时和8小时,检测MAPK的激活。我们应用MAPK的抑制剂以及MAPK的小分子干扰RNA处理细胞,然后应用50ug/ml的ox-LDL刺激细胞8小时,收集细胞。(3)在正常培养基或者ox-LDL刺激条件下,辛伐他汀干预细胞进行相关检测。3.2动物模型的建立120只8周龄雄性ApoE-/-小鼠随机分为两组—普通饮食组(n=40)和高脂饮食组(n=80)。2周后,所有小鼠均给予右侧颈总动脉套管以促进AS斑块形成。套管术后6周,普通饮食组随机分为两组(每组20只)Mockl组(0.5%methylcellulose单独灌胃)和Sim1组(辛伐他汀溶于0.5%甲基纤维素,50mg/kg·d量灌胃)；高脂饮食组随机分为四组(每组20只)Mock2组(0.5%甲基纤维素单独灌胃),SB组(p38MAPK抑制剂SB203580,2mg/kg·d腹腔注射),PD组(ERK1/2抑制剂PD98059,2mg/kg·d腹腔注射),Sim2组(辛伐他汀溶于0.5%甲基纤维素,50mg/kg·d量灌胃)。治疗6周后对小鼠称重行安乐死。3.3RT-PCR收集细胞,提取mRNA,检测P4Hα1mRNA水平。3.4Western blot收集细胞和颈动脉斑块组织,提取蛋白,检测P4Hα1、I型胶原和Ⅲ型胶原、P-/T-p38MAPK、P-/T-JNK、P-/T-ERK1/2的表达。3.5ELISA收集细胞上清,ELISA试剂盒检测上清液中Ⅰ型和Ⅲ型胶原含量。3.6Dil-ox-LDL摄取实验DiI-ox-LDL刺激细胞8小时,激光共聚焦照相观察细胞摄取情况。3.7血脂检测动物试验结束前,收集小鼠空腹血清,检测总胆固醇(TC)、甘油三脂(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)的水平。3.8H&E、油红O、天狼猩红染色以及MOMA-2、α-SM actin和ox-LDL的免疫组化染色制备颈动脉斑块冰冻组织切片,行H&E染色观察大体形态,油红O染色检测脂质含量,天狼猩红染色检测胶原含量,MOMA-2和a-SM actin染色分别检测巨噬细胞和平滑肌细胞含量,计算易损指数。免疫组化染色检测斑块内ox-LDL的含量。4结果4.1细胞实验4.1.1ox-LDL对P4Hαl的抑制作用在时间-效应实验中,50ug/ml的ox-LDL能够抑制P4Hα1的表达,在8小时时间点达到最大抑制效应；在剂量-效应实验中,随着ox-LDL浓度升高,P4Ha1的mRNA和蛋白水平呈递减趋势,在50ug/ml达到最大抑制效应。我们也应用Western blot和ELISA检测了ox-LDL刺激细胞8小时后胶原的含量,结果显示ox-LDL显著抑制Ⅰ型和Ⅲ型胶原的表达和分泌。4.1.2ox-LDL通过激活p38MAPK和ERK1/2信号通路发挥抑制作用Ox-LDL刺激平滑肌细胞0分钟、5分钟、15分钟、30分钟、2小时、4小时和8小时,检测MAPK激活。结果发现p38MAPK和ERK1/2磷酸化水平在5分钟达高峰,之后虽然逐渐减弱,但在8小时仍显著高于0分钟时,说明ox-LDL对其激活可持续至8小时。但是,ox-LDL对JNK的磷酸化水平无明显影响。应用p38MAPK、JNK和ERK1/2的抑制剂或者siRNA处理细胞,然后应用ox-LDL刺激细胞检测P4Hal的表达,发现抑制或基因沉默p38MAPK和ERKl/2后ox-LDL对P4Hα1的抑制作用明显减弱,而JNK的阻断或者基因沉默对P4Hα1的表达无显著影响。4.1.3辛伐他汀逆转ox-LDL对P4Hα1的抑制作用辛伐他汀不能改变正常情况下平滑肌细胞P4Hα1的表达水平,但是可以显著逆转ox-LDL刺激细胞对P4Ha1的抑制效应,相应的增加了胶原的合成。探讨其机制发现辛伐他汀能够减少细胞对ox-LDL的摄取,抑制p38MAPK和ERK1/2的磷酸化。4.2动物实验4.2.1动物体重及血脂水平各组ApoE-/-小鼠的体重无显著差异。高脂饮食小鼠TC、TG、LDL-C较普通饮食小鼠显著升高而HDL-C显著降低；高脂饮食组内的四个亚组间各血脂指标无显著差异。4.2.2辛伐他汀、SB203580和PD98059增加AS斑块稳定性普通饮食组内,辛伐他汀干预未显著改变斑块的易损指数。与Mockl组相比,Mock2组AS斑块的易损指数显著升高,提示动脉粥样硬化易损斑块造模成功。在高脂饮食组内,辛伐他汀、SB203580和PD98059干预能够显著降低斑块的易损指数。4.2.3辛伐他汀上调不稳定斑块内P4Ha1与Mockl相比,Mock2组P4Ha1的表达水平显著降低,提示不稳定斑块内P4Ha1生成减少。在普通饮食组内,辛伐他汀干预未明显改变P4Ha1的表达。但是,在高脂饮食组内,辛伐他汀、SB203580和PD98059干预显著增加斑块内P4Hα1的表达水平。与Mock2组相比,辛伐他汀治疗还能够降低斑块内ox-LDL的含量,抑制p38MAPK和ERKl/2的磷酸化。5结论(1) ox-LDL通过激活p38MAPK和ERK1/2信号通路抑制P4Ha1和胶原的合成。(2)他汀类药物能够逆转ox-LDL对P4Hα1的抑制作用,其机制是他汀类药物减少了细胞对ox-LDL的摄取,抑制p38MAPK和ERKl/2的激活。(3)在ApoE-/-小鼠模型中,他汀类药物可增加AS斑块内P4Hα1的表达和胶原合成,达到稳定斑块的作用。本研究揭示了他汀类药物稳定斑块的新的干预靶点,具有重要的学术意义。1研究背景急性冠脉综合征(acute coronary syndrome, ACS)是引起急性心脑血管事件的主要原因。目前已证实动脉粥样硬化(atherosclerosis, AS)易损斑块破裂是ACS发病的始动环节。研究发现易损斑块具有以下特点：较大的脂核、较薄的纤维帽、多种炎症细胞聚集以及斑块内胶原合成降解失衡。其中,胶原代谢是影响斑块稳定性的重要原因。4羟基-脯氨酸羟化酶(P4H)是所有己知的21种胶原合成过程中的关键酶,能够催化X-脯氨酸-甘氨酸(X-Pro-Gly)重复序列中的脯氨酸变为羟基脯氨酸,从而促进前胶原多肽链折叠成稳定的三螺旋结构。作为P4H的同工酶,P4Hα1是胶原合成过程中的重要限速酶。抑制P4Hαt1的表达可减少AS斑块内胶原合成,进而导致易损斑块形成；与之相反,过表达P4Hα1则会促进胶原的合成。既往研究发现TGFβ1能够促进P4Hα1的表达而吸烟可抑制其表达。近年来,本实验室张澄等研究发现在人类主动脉平滑肌细胞中,TNF-a具有抑制P4Ha1表达的作用,并提出了完整的信号转导通路即ASK1-MKK4-JNK1-NonO,首次提出NonO是P4Hal合成的重要转录因子,在调节胶原合成过程中发挥重要作用,但是目前尚缺乏动物实验验证。NonO(即人类的p54nrb蛋白)是一个大小为54KDa并在体内广泛表达的蛋白,是一个无POU结构域的八聚体结合蛋白,NonO含有两个核酸结合域,可直接与DNA结合参与转录调控；另外,NonO也可与其他转录蛋白相互作用调节基因表达。如前所述,本实验室研究发现NonO是P4Hal合成的重要转录因子,在调节胶原合成过程中发挥重要作用。既往研究还发现NonO参与调节环磷酸腺苷(cyclic adenosine monophosphate, cAMP)信号通路,而该信号通路可参与炎症反应,促进TNF-α、IL-2、IL-6等炎症因子的表达；另外,NonO能够参与COX-23’-UTR的多聚腺苷酸化,影响其转录,而COX-2亦参与了炎症反应和AS的进展。上述研究提示NonO可能参与调节胶原代谢和炎症反应。综合国内外研究现状,NonO在AS中的作用研究尚未明确。对于大多数急性心血管事件来说,易损斑块的稳定性是影响事件的决定性因素,但目前,NonO是否影响易损斑块的稳定性尚无报道。2研究目的(1)体内实验中,通过过表达和基因沉默NonO观察NonO对易损斑块破裂率的影响以及斑块内成分的改变。(2)体外实验中,通过过表达和基因沉默NonO探讨NonO稳定斑块的分子机制。3方法3.1NonO干扰及过表达慢病毒载体的构建构建三个干扰(si-A、si-B、si-C)和一个过表达NonO的慢病毒载体,以携带乱序siRNA的慢病毒载体作为干扰对照,以只携带绿色荧光蛋白基因的慢病毒载体作为过表达对照。转染RAW264.7细胞筛选最佳的干扰序列以及验证携带NonO基因的慢病毒的过表达效果。3.2动物模型的建立(1)20只8周龄雄性ApoE-/-小鼠高脂喂养2周后,随机分为两组：Control组(Il=10)：无颈动脉套管及精神应激；Mock组忙=10)：行颈动脉套管手术,手术8周后行精神应激,具体方法如下：将ApoE-/-小鼠置于容积为50ml、带有通气孔的塑料针管中,同时进行噪音刺激。噪音刺激强度为110dB,每5分钟一次,每次持续3秒。每天持续6小时,共应激四周。实验第14周末,ApoE-/-小鼠被处以安乐死。(2)为观察慢病毒转染效率及时间变化,15只ApoE-/-小鼠行颈动脉套管手术后八周,颈动脉局部转染携带GFP基因的慢病毒,并分别于转染前(n=5)、转染pGC-GFP-LV后2周(n=)和转染pGC-GFP-LV后4周(n=5)进行取材,观察各个时间点慢病毒转染效率。(3)8周龄雄性ApoE-/-小鼠高脂喂养2周后,右侧颈总动脉套管诱导AS斑块形成。颈总动脉套管手术8周后,根据转染病毒的不同,将ApoE-/-小鼠随机分为5组：(1) Control组(不加干预)；(2) si-N.C组(转染携带乱序siRNA的慢病毒、；(3) si-NonO组(转染携带si-NonO的慢病毒)；(4)N.C组(转染只携带GFP的慢病毒)；(5)NonO组(转染携带NonO基因的慢病毒)。转染后第3天开始应激刺激,刺激方法同前。实验第14周末,ApoE-/-小鼠被处以安乐死。3.3体重及血脂的检测实验开始前和14周末对所有ApoE-/-小鼠行体重测量,每只小鼠测量3次后取其平均值。实验14周末穿刺左心室采血,常温静置30分钟以后置于离心机中3000转离心15分钟,取上层血清,酶法检测血清总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和高密度脂蛋白胆固醇(HDL-C)的浓度。3.4组织病理学的检测制备右侧颈总动脉切片,对颈总动脉斑块进行H&E、油红O和天狼猩红染色并应用免疫组织化学染色的方法检测颈总动脉斑块内巨噬细胞、平滑肌细胞、IL-1β、IL-6、MMP-2和MMP-9的含量,计算易损指数。3.5细胞培养(1)在时间梯度实验中,RAW264.7细胞应用100ng/ml的TNF-α分别刺激0、6、12、24和48h；在剂量梯度实验中,我们应用0、25、50和100ng/ml的TNF-α刺激RAW264.7细胞24h。收集细胞提取蛋白检测NonO表达水平。(2)根据转染病毒的不同,将细胞分为5组：①Control组：不加任何干预；②si-N.C组：转染携带乱序siRNA的慢病毒；③si-NonO组：转染携带si-NonO的慢病毒；④N.C组：转染只携带GFP的慢病毒；⑤NonO组：转染携带NonO基因的慢病毒。100ng/ml的TNF-α刺激24小时,收集细胞。3.6实时定量PCR收集右侧颈总动脉组织,提取mRNA,检测颈动脉斑块内NonO、MMP-2和MMP-9的mRNA水平。3.7Western blot收集右侧颈总动脉斑块组织和细胞,提取蛋白,检测颈动脉斑块内NonO. MMP-2、MMP-9、P4Hα1、LOX-1、COX-2以及平滑肌细胞内NonO、MMP-2.MMP-9、IL-1β、MCP-1、ICAM-1、VCAM-1、p/t-NF-κB p65以及IκBα的蛋白表达水平。3.8细胞免疫荧光化学细胞免疫荧光化学法检测五组RAW264.7细胞内NF-κB的核转位情况。3.9免疫共沉淀收集细胞蛋白,检测TNF-α刺激RAW264.7细胞后NonO与NF-κB的结合情况。4结果4.1AS斑块内以及TNF-α刺激的RAW264.7内NonO的表达水平在体内实验中,与Control组正常的颈动脉相比,Mock组小鼠颈动脉斑块内NonO mRNA及蛋白表达显著升高,提示NonO在斑块进展中可能发挥作用。在体外实验中,时间梯度实验显示100ng/ml TNF-α能上调NonO的表达,在24小时达到最大效应；浓度梯度实验显示10ng/ml TNF-α刺激24小时便能显著提高NonO的表达,在100ng/ml时达最大效应。4.2RAW264.7细胞中慢病毒干扰及过表达NonO的效率和效果将N.C、si-A、si-B、si-C及NonO-LV分别转染小鼠RAW264.7细胞,转染4天后观察慢病毒携带的报告基因GFP的表达情况,发现感染效率均大于70%进而收集细胞提取蛋白质进行检测。结果表明,si-A、si-B、si-C转染致NonO蛋白表达分别减少57%、43%和44%,进而筛选出最有效的干扰位点si-A用于随后动物和细胞实验中si-NonO组的转染；而NonO-LV转染细胞能够使NonO蛋白水平较Control组增加70%,用于随后动物和细胞实验中NonO组的转染。4.3慢病毒在颈动脉斑块的转染效率在颈动脉斑块局部转染慢病毒开始前、2周末和4周末取材,发现转染两周后GFP显著表达,然后开始衰减,4周末斑块内仍可见GFP表达,但是较2周前已有减弱。4.4颈动脉斑块内NonO干扰及过表达效果留取颈动脉斑块组织,检测斑块内NonO的mRNA和蛋白表达水平。结果显示,si-NonO组NonO的]mRNA和蛋白水平较Control组分别下降了47%和43%,而NonO组则分别增长了61%和59%,但在si-N.C组和N.C组中NonO的mRNA和蛋白水平与Control组相比无显著差异。4.5实验动物一般情况实验各组ApoE-/-小鼠均顺利完成实验,慢病毒转染小鼠中未观察到明显的局部和全身性的不良反应。各组ApoE-/-小鼠的体重无显著性差异,血清中TC、 TG、LDL-C和HDL-C的浓度亦无发现显著统计学差异。4.6NonO对AS斑块破裂率的影响通过对连续组织病理切片的H&E染色分析,我们发现Control组、si-N.C组和N.C组各有46.67%(7/15),si-NonO组有13.33%(2/15),NonO组有66.67%(10/15)的AS斑块发生破裂。统计分析显示,si-NonO组斑块破裂率显著低于Control组,而NonO组斑块破裂率则显著高于Control组;Control组、si-N.C组和N.C组三组间斑块破裂率无统计学差异。含铁血黄素染色进一步验证了斑块破裂。4.7NonO对AS斑块成分及其易损性的影响与Control组相比,si-NonO组的斑块内巨噬细胞含量、脂质含量和易损指数显著降低,而平滑肌细胞含量和胶原含量显著升高；NonO组结果与si-NonO组相反。Control组、si-N.C组和N.C组间上述指标无显著差异。4.8NonO对P4Hα1表达的影响体内实验显示,Control组、si-N.C组和N.C组三组间颈动脉斑块内P4Hα1的蛋白表达无显著差异。与Control组相比,si-NonO组颈动脉斑块内P4Hα1的蛋白水平显著增高,而NonO组则显著降低,说明NonO参与抑制P4Hα1的表达。4.9NonO对基质金属蛋白酶表达的影响在体内实验中,si-NonO组颈动脉斑块内MMP-2和MMP-9的mRNA水平显著低于Control组；免疫组化和western blot检测也证明MMP-2和MMP-9蛋白水平在si-NonO组显著降低,而NonO组MMP-2和MMP-9mRNA和蛋白水平较Contro组显著升高。Control组、si-N.C组和N.C组三组间MMP-2和MMP-9表达水平无显著差异。体外Western blot结果与该结果一致。同时,我们也检测了RAW264.7细胞刺激后MMP-2和MMP-9活性的改变,结果发现与Control组相比,si-NonO组MMP-2和MMP-9活性均显著降低,而NonO组则显著升高。4.10NonO对炎症因子的影响在体内实验中,免疫组化分析显示si-NonO组的炎症因子IL-1β和IL-6的蛋白水平较Control组显著降低,而NonO组则显著增加IL-1p和IL-6的蛋白表达；western blot结果显示si-NonO组的LOX-1和COX-2的蛋白表达显著低于Control组,而NonO组LOX-1和COX-2的蛋白表达则显著升高。在体外实验中,Western blot结果显示与Control组相比,si-NonO组IL-1β、MCP-1、ICAM-1和VCAM-1均显著降低,而NonO组则显著升高。4.11NonO与NF-κB的相互作用免疫共沉淀检测发现TNF-α能够增加NonO与NF-κB的结合。细胞免疫荧光染色显示与Control组相比,si-NonO组NF-κB的核转位显著降低,而NonO组NF-κB的核转位显著增强。Western blot检测显示si-NonO组的NF-κB磷酸化水平较Control组显著降低,而NonO组显著升高；IκBα的变化趋势与NF-κB磷酸化水平的变化趋势相反。5结论(1)首次发现NonO在动脉粥样硬化硬化斑块中高表达。(2)在ApoE-/-小鼠易损斑块模型中,NonO降低斑块稳定性,增加斑块的破裂率,而NonO基因沉默后斑块稳定性增强,破裂率降低。(3) NonO降低斑块稳定性的机制为增加MMP-2和MMP-9的生成,减少P4Ha1的表达以及抑制NF-κB介导的局部炎症反应。1研究背景动脉粥样硬化(atherosclerosis, AS)是一组易感基因和危险因素共同作用导致的血管疾病。糖尿病(dibetes mellitus, DM)是AS最重要的危险因素之一。鉴于DM在AS中的重要作用,DM己作为冠状动脉粥样硬化性心脏病的等危症。DM可引起心肌梗死和脑卒中等大血管疾病,也可引起慢性肾病即糖尿病肾病(Diabetic nephropathy, DN)和神经病变等微血管疾病。多项大规模的临床试验结果表明,积极控制糖尿病患者的血糖水平有助于减轻微血管病变,但无助于改善大血管病变,提示两种血管病变的发生机制可能不同。因此,进一步探讨糖尿病微血管病变的发生机制和干预靶点具有重要的临床意义。由于DN是公认的糖尿病微血管病变,因此本文选择DN作为研究对象。研究发现肾素-血管紧张素系统(Renin-angiotensin system, RAS)在DN的发病过程中起了关键作用,应用ACEI/ARB可有效的缓解糖尿病的肾脏损伤,但是并不能达到预期效果,因此,寻找RAS系统新的干预靶点以有效缓解DN成为亟待解决的问题。ACE2是ACE的同源物,本实验室研究发现ACE2及其产物Ang(1-7)能有效阻止AS的发生发展；由于糖尿病是冠心病等危症,我们因此又应用外源性ACE2干预糖尿病大鼠模型,发现ACE2也显著改善了DM心脏功能并减轻了糖尿病肾脏损伤。目前已有大量的证据证实ACE2能够延缓DN的发展,然而,作为ACE2的主要产物,Ang(1-7)在DN中的独立作用仍不明确。因此,深入研究其在DN中的作用对于认识DN的发病机制以及DN的治疗具有重要意义。Ang(1-7)是一种7肽,是RAS系统中一种重要产物。在肾脏组织内,Ang(1-7)主要由ACE2水解AngII生成,在维持肾脏内环境稳定中发挥重要作用。AngII可通过多种机制促进DN的发展,包括升高全身血压以及肾内血流灌注压,通过激活TGF-β、VEGF、内皮素等促进细胞外基质的合成以及刺激氧化应激等等。而Ang(1-7)是一种舒血管肽,可以对抗AngII的有害作用；同时Ang(1-7)可以减轻NADPH氧化酶(NADPH oxidase, NOX)介导的氧化应激从而减轻蛋白尿并改善血管功能；另外,研究发现Ang(1-7)还能够抑制TGF-p的生成。Ang(1-7)的上述效应可能会延缓DN的发展。近期研究还发现给予糖尿病小鼠重组人类ACE2能够通过减少NOX活性和PKCα、PKCβ1的生成减轻DN的进展,这种保护作用可能是由AngII水平减低和Ang(1-7)生成增加介导的。研究发现外源性的Ang(1-7)的应用能够明显减轻STZ诱导的糖尿病大鼠的蛋白尿、肾内胶原成分并能改善内皮功能,其原因可能是Mas受体的激活。然而,Shao Y等却发现了与其上完全相反的一个现象：Ang(1-7)明显加重STZ诱导的糖尿病大鼠的肾功损伤。如前所述,目前关于外源性Ang(1-7)对DN作用的研究并不多,而且以上研究还存在以下几个重要问题亟待解决：1、目前,Ang(1-7)对于DN的确切作用是有益还是有害仍存有争议；2、不同剂量的Ang(1-7)对DN的具体作用尚未见报道；3、Ang(1-7)与ARB联合治疗是否优于单一治疗目前仍不清楚。2研究目的(1)通过STZ注射构建DN的大鼠模型,观察Ang(1-7)对DN的作用,同时探讨Ang(1-7)与ARB联合应用对DN的治疗效果。(2)体外实验中探讨Ang(1-7)对DN作用的具体机制,为抗DN治疗提供新的思路和药物作用的新靶点。3方法3.1动物模型120只10周龄的雄性Wistar大鼠随机分为两组：Control组(n=15,腹腔注射生理盐水)和DM组(n=105,腹腔注射STZ溶液,65mg/kg)。STZ注射48小时后,检测DM组大鼠尾静脉血糖水平≥16.7mmol/l为造模成功。DM大鼠每3天腹腔注射一次胰岛素(2-3U)维持血糖在16.7-25mmol/l以防止高糖诱导的死亡。12周后,DM大鼠随机分为以下7组(每组15只)： NT组：不接受任何药物干预；S-Ang(1-7)组：200ng/kg-min的Ang(1-7)；M-Ang(1-7)组：400ng/kg·min的Ang(1-7); L-Ang(1-7)组：800ng/kg-min的Ang(1-7)；Valsartan组：30mg/kg·d的缬沙坦,灌胃给药；L+V组：800ng/kg-min的Ang(1-7)+30mg/kg-d的缬沙坦；L+A779组：800ng/kg·min的Ang(1-7)+800ng/kg-min的A779。Ang(1-7)和A779均采用植入式胶囊渗透压泵皮下包埋持续泵入的给药方式。药物干预前收集大鼠尿液并颈静脉穿刺收集空腹静脉血。药物干预4周,收集大鼠24小时尿液及空腹血液。3.2大鼠一般情况检测实验结束后,测量各组大鼠的血糖、收缩压(SBP)、体重、肾重、24小时尿量、血液和尿液中肌酐含量并计算肌酐清除率。3.3ELISAELISA检测血清和尿液中的Ang(1-7)以及尿蛋白水平。3.4SOD和MDA测量分离肾小球,制备肾小球匀浆,检测肾小球组织SOD及MDA含量。3.5组织病理学检测大鼠肾脏组织切片,PAS染色计算肾小球硬化指数(GSI)。免疫组织化学染色检测肾小球内Ⅳ型胶原、TGF-β1、VEGF、PCNA和巨噬细胞含量。3.6细胞培养大鼠肾小球系膜细胞系(HBZY-1)分组：Control组：应用含5mmol/1葡萄糖的培养基培养,并加入20mmol/1的甘露醇以平衡渗透压；HG组：25mmol/1的葡萄糖刺激24小时；S-Ang(1-7)组、M-Ang(1-7)组和L-Ang(1-7)组：分别应用50、100和200nmol/1的Ang(1-7)预处理细胞1小时；Valsartan组：10-6mol/1的缬沙坦预处理细胞1小时；L+V组：200nmol/1Ang(1-7)+10-6mol/l缬沙坦预处理细胞1小时；L+A779组：200nmol/1A779预处理细胞半小时后加入200nmol/1Ang(1-7)处理1小时。所有药物干预组细胞在药物预处理后应用25mmol/1的葡萄糖刺激24小时,收集细胞。3.7Western blot分离并提取肾小球蛋白,western blot检测NOX4、p47phox、PKCα、PKCβ1、 TGF-β1和p-Smad3的蛋白表达水平。提取大鼠肾小球系膜细胞系HBZY-1的蛋白,western blot检测NOX4、p47phox、TGF-β1、p-Smad3、VEGF和IV型胶原。试剂盒分离并提取HBZY-1细胞的膜蛋白和胞浆蛋白,western blot分别检测两者的PKCa和PKCβ1的蛋白水平。3.8DHE、DCF和EdU荧光染色各组HBZY-1细胞在实验结束时应用DHE和DCF染色检测ROS水平。EdU染色检测细胞增殖水平。4结果4.1血清和尿液中Ang(1-7)水平Ang(1-7)干预治疗前,DM大鼠各组的血清和尿液Ang(1-7)水平显著低于Control组。但是Ang(1-7)干预后,血清和尿液中Ang(1-7)水平呈剂量依赖性升高。另外与NT组相比,缬沙坦组也能升高Ang(1-7)的含量。4.2血压和血糖检测DM大鼠的SBP较Control组显著降低,而DM大鼠各组间无显著性差异。DM大鼠血糖水平显著高于对照组,DM大鼠组间无显著性差异。4.3Ang(1-7)改善肾脏功能与Control组相比,体重和肌酐清除率在NT组显著降低而肾重／体重、24小时尿量、血清肌酐水平和24小时尿蛋白量在NT组却显著升高,提示DN造模成功。Ang1-7)、缬沙坦以及两者联合治疗均能改善上述指标。Ang(1-7)的效应呈剂量依赖性,而A779能阻断Ang(1-7)的上述作用。L-Ang(1-7)组和L+V组肾脏功能改善效果相似并且均优于Valsartan组。4.4Ang(1-7)减轻肾小球硬化PAS染色并根据其阳性面积计算GSI。结果发现,DM各组的GSI均显著高于Control组。与NT组相比,Ang(1-7)能剂量依赖性的降低GSI,而A779则能够阻断该作用。L-Ang(1-7)组与L+V组组间GSI无显著性差异,但是此两组的GSI水平均显著低于Valsartan组。免疫组化显示NT组肾小球内IV型胶原、TGF-β1、VEGF和PCNA含量均显著高于Control组。Ang(1-7)、Valsartan以及两者联合治疗均能降低上述指标,而且Ang(1-7)的治疗效应呈剂量依赖性,A779能阻断其作用。与Valsartan组相比,L-Ang(l-7)和L+V两组降低上述指标的作用更为明显,且两组间无显著差异。另外,肾小球内巨噬细胞浸润程度在各组间无显著性差异。4.5Ang(1-7)减少氧化应激体内实验中,与Control组相比,NT组的MDA含量显著增加,而SOD活性则显著降低。Ang(1-7)、缬沙坦以及两者联合治疗均能减少MDA含量而增加SOD活性,其中Ang(1-7)的效应呈剂量依赖性,而A779能阻断该效应。L-Ang(1-7)组与L+V组治疗效果相似,且均优于Valsartan组。在体外实验中,我们应用DHE和DCF染色观察HBZY-1细胞内ROS含量。结果发现,HG组ROS含量显著高于Control组,而Ang(1-7)能减少ROS含量,在L-Ang(l-7)组达到最大效应。L-Ang(1-7)和L+V两组间ROS含量无显著差异,但是均低于Valsartan组。4.6Ang(1-7)减少NOX和PKC的表达提取肾小球蛋白,western blot检测显示NT组NOX4、p47phox、PKCβ1显著高于Control组,而Ang(1-7)剂量依赖性减少上述指标的表达,其大剂量的治疗作用与Ang(1-7)联合缬沙坦治疗作用相似,并且优于单纯缬沙坦治疗,A779治疗能阻断Ang(1-7)的作用。体外实验中Ang(1-7)对NOX4和p47phox的作用与动物实验一致。另外,我们分离了HBZY-1细胞膜蛋白和浆蛋白分别检测PKCa和PKCβ1在其中的含量,结果发现在浆蛋白中PKCa和PKCβ1在各组间无显著性差异,而在膜蛋白中,PKCα和PKCβ1的变化趋势与动物实验结果一致。4.7Ang(1-7)减少TGFβ1Smad3和VEGF信号通路以及IV型胶原的表达体内体外实验均发现,与Control组相比,NT组的TGFβ1蛋白表达和Smad3磷酸化水平均显著升高。Ang(1-7)、Valsartan和两者联合治疗均能抑制TGFβ1蛋白表达和Smad3的磷酸化水平,且Ang(1-7)的效应呈剂量依赖性,而A779能够阻断其作用。L-Ang(l-7)组和L+V两组的抑制作用强于Valsartan组,而两组间无显著性差异。我们又检测了HBZY-1细胞中VEGF和Ⅳ型胶原的蛋白表达,发现其变化趋势与TGFβ1的变化趋势一致。4.8Ang(1-7)减少肾小球系膜细胞的增殖EdU染色观察各组细胞间增殖水平,结果发现与Control组相比,HG组的EdU阳性细胞百分比显著升高。Ang(1-7)、Valsartan和两者联合治疗均能减少EdU阳性细胞比例,且Ang(1-7)的效应呈剂量依赖性,而A779能够阻断其作用。L-Ang(1-7)和L+V两组的抑制作用强于Valsartan组,而两组间无显著性差异。5结论(1)Ang(1-7)剂量依赖性改善STZ诱导的糖尿病肾病,大剂量Ang(1-7)的肾脏保护作用优于缬沙坦治疗。两药联合应用未发现明显协同效应。(2)Ang(1-7)介导的肾脏保护机制包括Ang(1-7)减轻NOXs和PKCs介导的氧化应激并抑制TGFβ1Smad3和VEGF信号通路。更多还原

【Abstract】 1IntroductionAtherosclerosis (AS) damages not only the large and medium-sized arteries, but also the small artery and arteriole, which leads to morphological and functional abnormalities of multiple organs. Heart, brain and kidney are the main target organs of AS, and AS could cause myocardial infarction, ischemic cardiomyopathy, stroke and chronic kidney diseases, finally resulting in multiple organ failure and death. Therefore, it is of important significance in theory and practice to study mechanisms and intervention targets of macrovascular and micro vascular lesions of AS.Atherosclerotic plaque rupture is the major cause of acute coronary syndrome (ACS) that leads to unstable angina, acute myocardial infarction and sudden death. Pathologically, plaques vulnerable to rupture are characteristic of a large lipid core and a thin fibrous cap. As collagen is the major component of fibrous caps, the content of collagen in plaques determines plaque vulnerability. Prolyl-4-Hydroxylase (P4H) is one of the key enzymes essential for the synthesis of all known types of collagen. P4H catalyzes proline located in repeating X-Pro-Gly triplets to hydroxyproline during posttranslational processing of collagen production. As an isoenzyme of P4H, P4Hal, a rate-limiting enzyme that folds the procollagen polypeptide chains into stable triple helical molecules, which is essential for collagen maturation and secretion. The low expression of P4Hal leads to reduce the content of collagen and then leads to the unstability of AS plaque. Previous studies found that upregulated the expression of P4Hα1whereas smoking induced P4Hα1degradation. Our studies also found that TNF-a and IL-6suppressed P4Hal expression via ASK1-JNK-NonO pathway and ERK1/2-Spl pathway, respectively.It is known that oxidized low density lipoprotein (ox-LDL) played the key role in the development of AS. Previous studies found that ox-LDL may. induce plaque instability by increasing lipid accumulation, initiating oxidative stress, and activating matrix metalloproteinases (MMPs). However, the direct effects of ox-LDL on P4Hα1expression and collagen synthesis is unclear. Statins have become the drug of first choice for the primary and secondary prevention of atherosclerotic disease. A wealth of evidence indicates that statins have pleiotropic effects such as lowered serum lipid level, improved endothelial function, reduced local inflammation and inhibited atherothrombosis. Previous studies reported that statins enhanced the collagen content in plaques by decreasing MMPs expression and increasing tissue inhibitors of metalloproteinase-1(TIMP-1) expression. However, the direct effects of statins on P4Hal expression and collagen production are unknown.2Objectives(1) To investigate whether ox-LDL suppresses the expression of P4Hα1and elucidate the underlying mechanisms and to detect the effect of simvastatin on it;(2) To investigate the effect of simvastatin on the expression of P4Hal and the underlying mechanisms and to explore a novel target of statins on stabilizing plaques.3Methods3.1Cell culture and treatment(1) To study the time-response of P4Hα1expression after ox-LDL stimulation, human aortic smooth muscle cells (HASMCs) were treated with50ug/ml ox-LDL at0h and fresh medium containing50ug/ml ox-LDL was used every8h till24h. The mRNA and protein expression of P4Hα1expression was assayed at0,4,8,12and24h after ox-LDL stimulation. To study the dose-response of P4Hal expression after ox-LDL stimulation, HASMCs were stimulated with0,25and50ug/ml ox-LDL for8h, respectively.(2) To examine the role of MAPK pathways in ox-LDL-mediated effects on P4Hα1expression, HASMCs pretreated with or without simvastatin for lh were stimulated with50ug/ml ox-LDL for0,5,15,30min and2,4,8h, and then the phosphorylation levels of p38MAPK, JNK and ERK1/2were assayed by western blot. Then, HASMCs were treated with the inhibitors or siRNA of p38MAPK, JNK and ERK1/2and then were incubated with ox-LDL for8h before cells were harvested for measurement.(3) To further study the effects of simvastatin on the impact of ox-LDL on P4Hal expression, HASMCs were cultured with or without50ug/ml ox-LDL for8h after pretreatment with or without10μmol/1simvastatin for1h. And then cells were harvested for measurement.3.2Animal modelOne hundred and twenty male ApoE-/-mice on a C57BL/6background (8weeks old) were randomly divided into two groups:a normal diet group (n=40) fed with a normal chaw and a high-fat diet group (n=80) fed with a diet containing0.25%cholesterol and15%cocoa butter. Two weeks later, a constrictive silastic tube was placed on the right common carotid artery in all mice after anesthesia. Six weeks after the surgery, mice in the normal diet group were randomly divided into two subgroups (n=20mice in each group):Mockl group that received oral methylcellulose alone, and Siml group that received an intragastric administration of50mg/kg·d simvastatin in0.5%methylcellulose. Mice in the high-fat diet group were randomly divided into four subgroups (n=20in each group):Mock2group that received oral methylcellulose alone, SB group that received an intraperitoneal injection of2mg/kg·d SB203580, PD group that received an intraperitoneal injection of2mg/kg-d PD98059, and Sim2group that received an intragastric administration of50mg/kg-d simvastatin. At the end of14weeks, all the mice were weighted and euthanized.3.3Quantitative real-time PCRTotal RNA was extracted from HASMCs to detect the expression level of P4Hα1mRNA.3.4Western blot analysisTotal proteins were extracted from HASMCs or the right common carotid arteries of ApoE-/-mice. The protein expression of P4Hal, type I and type III collagen, P-/T-p38MAPK, P-/T-JNK, P-/T-ERK1/2were analyzed by western blot.3.5ELISAThe content of soluble type I and III collagen in culture supernatants was assayed by ELISA.3.6Dil-ox-LDL uptakeHASMCs pretreated with or without simvastatin for1h were stimulated with50ug/ml Dil-ox-LDL for8h. After fixed and DAPI staining, the cells were placed for confocal microscopy.3.7Serum lipidsAt the end of the experiment, blood samples were collected by cardiac puncture in the mice fasted overnight to measure the serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) by an enzymatic assay.3.8Histopathological analysisSuccessive transverse cryosections were cut at5μm thickness and selectively stained with hematoxylin and eosin (H&E) at50μm intervals to select the point of maximal stenosis of arteries used for morphological analysis. Sirius red and Oil-red O staining were also performed. Frozen sections were stained for immunohistochemical analysis including macrophages (MOMA-2), SMCs (a-smooth muscle actin) and ox-LDL. The vulnerability index was calculated as (macrophage%+lipid%)/(collagen%+a-SM actin%).4Results4.1The in vitro experiment4.1.1ox-LDL-mediated suppression of P4Hal expressionIn the in vitro time-response study, ox-LDL suppressed the mRNA and protein expression of P4Hal significantly and the peak effect occurred after ox-LDL stimulation for8hours. In the in vitro dose-response study, ox-LDL also suppressed the expression of P4Hα1significantly and the peak effect was observed when HASMCs were treated with50ug/ml ox-LDL. Ox-LDL treatment also significantly reduced the expression levels of type I and III collagen in HASMCs and ELISA showed that ox-LDL also significantly reduced the protein levels of type I and III collagen in cell culture medium.4.1.2p38MAPK and ERKl/2pathways were involved in ox-LDL-induced downregulation of P4Hal expressionox-LDL induced the phosphorylation of p38MAPK and ERKl/2with the effect peaking at5min and lasting for8h. However, ox-LDL had no effect on the phosphorylation levels of JNK. To validate these results, HASMCs were pretreated with the inhibitors or siRNAs of p38MAPK, JNK and ERKl/2respectively before ox-LDL treatment. As a result, blockade or gene silencing of p38MAPK and ERKl/2attenuated ox-LDL-induced downregulation of P4Hal expression, whereas blockade or gene silencing of JNK had no effect on P4Hα1expression.4.1.3Simvastatin attenuated the suppressive effect of ox-LDL on P4Hα1expressionSimvastatin significantly inhibited the suppressive effect of ox-LDL on P4Hal expression, furthermore, simvastatin also largely reversed ox-LDL-induced reduction of type I and III collagen expression. However, simvastatin treatment alone without ox-LDL stimulation had no effect on P4Hal and collagen expression. Furthermore, simvastatin substantially reduced the phosphorylation levels of p38MAPK and ERKl/2in ox-LDL-stimulated HASMCs but had no effect on the phosphorylation of JNK. Furthermore, we found that simvastatin significantly decreased ox-LDL accumulation in the cells, suggesting that simvastatin inhibits the activation of p38and ERK probably via inhibiting ox-LDL uptake by HASMCs.4.2Animal model4.2.1Body weight and serum lipids in miceAt the end of the animal experiment, there were no significant difference in body weight among the six subgroups of mice. The serum levels of TC, TG and LDL-C increased while HDL-C decreased dramatically in the four subgroups of mice fed with a high diet relative to the Mockl subgroup but these serum lipid parameters did not differ among subgroups of mice fed with a high fat diet or with a normal diet. 4.2.2Simvastatin and p38MAPK and ERKl/2inhibitors enhanced plaque stabilityIn the normal diet group, there was no significant difference in vulnerability index between mice treated with and without simvastatin. The vulnerability index in mice fed with a high-fat diet only (Mock2group) was significantly higher than that in Mockl subgroup, indicating that high-fat diet feeding alone may increase plaque vulnerability in ApoE-/-mice. We also found that the vulnerability index was significantly decreased in mice receiving both high-fat diet and treatment with simvastatin or inhibitors of p38MAPK and ERKl/2, compared with the Mock2subgroup. These results demonstrated that simvastatin and p38and ERK1/2inhibitors had a plaque stabilizing effect in apoE-/-mice.4.2.3Simvastatin upregulated P4Hal expression in carotid plaquesThe levels of p38and ERK1/2phosphorylation and P4Hα1protein expression showed no significant difference between the two subgroups in mice fed with a normal diet. In contrast, the protein expression level of P4Hal was substantially decreased whereas the phosphorylation levels of p38and ERKl/2were significantly increased in the Mock2subgroup relative to the Mockl subgroup. The protein expression levels of P4Hα1and the content of collagen in the carotid plaques were significantly increased whereas the phosphorylation levels of p38and ERK1/2were reduced in mice receiving both a high-fat diet and treatment with simvastatin or inhibitors of p38MAPK and ERKl/2, in comparison with the Mock2subgroup. Furthermore, simvastatin treatment significantly reduced the relative content of ox-LDL in plaques relative to the Mock2subgroup. These results showed that simvastatin upregulated P4Hal expression by inhibiting ox-LDL uptake and inactivating p38and ERK1/2pathways in the carotid plaques.5Conclusion(1) Ox-LDL suppressed the expression of P4Hal and collagen via activating p38MAPK and ERK1/2signaling pathway.(2) Statins attenuated the suppressive effect of ox-LDL on P4Hal.The underlying mechanisms were that statins decreased the uptake of ox-LDL in HASMCs and then suppressed the activation of p38MAPK and ERKl/2.(3) In ApoE-/- mice, statins increased the expression of P4Hal and the content of collagen in AS plaques and increased the plaque stability. 1IntroductionAtherosclerotic plaque vulnerable to rupture is the major cause of acute coronary syndrome (ACS), which is identified by a thin, weakened fibrous cap, a large lipid core, accumulation of inflammatory cells and the imbalance between extracellular matrix (ECM) synthesis and degradation. As collagens are the major component of the fibrous cap of atherosclerotic plaques, the strength of plaque depends on a dynamic balance of collagen synthesis and degradation.Prolyl-4-Hydroxylase (P4H) is one of the key enzymes essential for the synthesis of all known types of collagen. P4H could catalyze proline located in repeating X-Pro-Gly triplets to hydroxyprolin which folds the procollagen polypeptide chains into stable triple helical molecules. As an isoenzyme of P4H, P4Hα1, is rate-limiting and essential for collagen maturation and secretion. The suppressed expression of P4Hal led to the decreased content of collagen in AS plaques. On the contrary, overexpression of P4Hal led to collagen synthesis.NonO, originally identified as a non-POU-domain-containing, octamer-binding protein, is55kDa ubiquitously expressed protein which contains two kinds of nucleic acid binding domains which could bind to both DNA and RNA. NonO could regulate the expression of many genes not only through directly binding to DNA as a transcriptional factor but also through binding to other protein as a cofactor. So, NonO could regulate many genes through different ways, among which some genes are in close relationship with atherosclerosis. NonO acts as a component of the cAMP-signaling pathway and is necessary for the activation of cAMP response element binding protein (CREB) target genes which contains TNF-a, IL-2and IL-6, and so on, suggesting that NonO may participate in promoting inflammation. NonO can bind to the auxiliary upstream sequence elements of cyclooxygenase-2(COX-2) and regulate its expression which also participate in the progression of AS. So, these studies suggests that NonO may take part in inflammation and atherosclerosis. Especially in recent years, Zhang C et al found that in HASMCs, NonO silencing dramatically attenuated TNF-a suppression of P4Hal gene expression and collagen synthesis. However, the exact role of NonO in vulnerable plaque is unclear.2Objectives(1) In the in vivo experiments, we delivered NonO-lentivirus (LV) or si-NonO-LV into the carotid plaques of apolipoprotein E-deficient (ApoE-/-) mice to detect its role in plaque disruption and plaque composition.(2) To elucidate the underlying molecular mechanisms of NonO-mediated detrimental effects on vulnerable plaque.3Methods3.1Preparation of Lentiviral vectors and Target screening of siRNANonO-overexpression lentivirus (NonO-LV) and three si-NonO lentivirus (si-A, si-B, si-C) were transduced into RAW264.7cells at a multiplicity of infection (MOI) of40.3days after transduction, cells were harvested for western blot to detect the overexpression effect and screen the most effective siRNA of NonO.3.2Animal model(1)20male ApoE-/-mice (8weeks of age) were fed with a high-fat diet (0.25%cholesterol and15%cocoa butter) for2weeks, and then randomly divided into two groups:Control group (n=10) which got no surgery; Mock group (n=10) which were placed a constrictive silastic tube around the right common carotid artery.8weeks later, mice in Mock group were restrained in a50-ml plastic tube with multiple holes on the wall to ensure sufficient ventilation and sound transmission, and then the tubes were put into a noise generator that emits noise every5min at110dB for3sec. The noise and restraint stress lasted for6h per day for4weeks. At the end of14weeks, all the mice were euthanized.(2)15male ApoE-/-mice were placed a constrictive silastic tube around the right common carotid artery.8weeks after surgery, the mice were transfected with lentivirus and euthanized before transfection (n=5),2weeks after transfection (n=5) and4weeks after transfection (n=5) to detect the tansfection efficiency of lentivirus.(3)125male ApoE-/-mice (8weeks of age) were fed with a high-fat diet for2weeks. Then all the mice were placed a constrictive silastic tube on the right common carotid artery to induce atherosclerotic lesion as described previously.8weeks after surgery, all the mice were divided into5groups (n=25per group) named:Control group, si-N.C group, si-NonO group, N.C group and NonO group, respectively, which were delivered to plaques with physiological saline, siRNA-N.C-LV, si-NonO-LV, pGC-GFP-LV and NonO-LV respectively as previously described.3days after lentiviral transfection, all the mice underwent stress stimulation as mentioned above. At the end of14weeks, all the apoE-/-mice were euthanized to collect the right common carotid arteries and blood from left ventricular for further analysis.3.3Body weight and Serum lipid profileBefore and at the end of the experiment, body weight of all the mice was measured. The serum concentrations of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were measured by an enzymatic assay.3.4Histopathological analysisSerial cryosections of the carotid arteries were stained by H&E, oil-red O, sirius red. In addition, frozen sections were stained for immunohistochemical analysis included macrophages, SMCs, MMP-2, MMP-9, IL-1β,IL-6. The vulnerable index was calculated by the following formula:the relative positive staining area of (macrophages%+lipid%)/the relative positive staining area of (α-SMCs%+collagen%).3.5Cell treatment(1) For the time course study, we treated RAW264.7cells with100ng/ml TNF-a for0,6,12,24and48h. For the dose-dependent effects, we treated RAW264.7cells with0,25,50and100ng/ml for24h. All the cells were harvested for measurement of protein expression.(2) RAW264.7cells were cultured in6-well plates (2x106) in DMEM supplemented with10%FBS without antibiotics. Overnight after plating, cells were divided into five groups:①Control group:cells without transfection;②si-N.C group:cells transfected with siRNA-N.C-LV;③si-NonO group:cells transfected with si-NonO-LV;④N.C group:cells transfected with pGC-GFP-LV;⑤NonO-LV group:cells transfected with NonO-LV.3days after transduction, all the cells were stimulated with100ng/ml TNF-a for24h and then harvested for further analysis.3.6Quantitative real-time PCRTotal mRNA was extracted from the right common carotid arteries of ApoE-/-mice to detect the expression levels of NonO, MMP-2and MMP-9.3.7Western blotTotal proteins were extracted from the right common carotid arteries and RAW264.7cells. The protein expression levels of NonO, MMP-2, MMP-9, P4Hα1, LOX-1and COX-2in carotid plaques and NonO, MMP-2, MMP-9, IL-1β, MCP-1, ICAM-1, VCAM-1, p/t-NF-KB and IκBα in RAW264.7cells were detected by western blot.3.8Co-immunoprecipitationCo-immunoprecipitation assays were performed in RAW264.7cells treated with or without TNF-a to detect the combination of NonO and NF-κB.3.9ImmunofluorescenceImmunofluorescence were performed in RAW264.7cells to detect the effect of NonO on NF-κB p65nuclear translocation.4Results4.1Upregulation of NonO expression in atherosclerotic plaque and TNF-a-induced RAW264.7cellsIn ApoE-/-mice, compared with the control group, both mRNA and protein expression of NonO in Mock group were significantly increased, which suggested that a potential role of NonO in the pathogenesis of atherosclerosis.We also examined NonO expression in TNF-α-stimulated RAW264.7cells. In the time-response study, TNF-α upregulated the protein expression of NonO and the peak effect occurred at24h. In the dose-response study, TNF-α also increased the protein expression of NonO and the peak effect was observed when RAW264.7cells were treated with100ng/ml TNF-α.4.2Efficiency and effect of lentiviral transfection in vitroMice macrophage RAW264.7cells were transfected with three si-NonO-LVs (si-A, B and C) and the NonO-LV to detect their effects. Si-A, B and C exhibited57%,43%and44%reduction, respectively, in NonO protein expression. So, si-A were selected for further studies. NonO-LV increased the protein expression level of NonO by70%. So, si-NonO-LV and NonO-LV effectively knockdown and overexpressed the expression of NonO, respectively.4.3Efficiency of lentiviral transfection in vivoSince GFP expression provides a convenient monitor for detecting the transfection efficiency of lentivirus, the GFP fluorescence in the plaques was examined in0,2and4weeks after transfection.2weeks after transfection, carotid artery plaques had the obvious GFP fluorescence, which suggested a successful transfection of lentivirus.4weeks after transfection, GFP fluorescence was still visible in plaques, although their densities became weak. These results showed that lentivirus was efficiently transfected into atherosclerotic plaques.4.4The effect of NonO silencing and overexpression in carotid plaquesAt the end of our study, the expression levels of mRNA and protein of NonO in the carotid arterial plaques were detected by RT-PCR and western blot. Compared with them in control group, the levels of mRNA and protein of NonO were significantly decreased in si-NonO group by about47%and43%, respectively and increased in NonO-LV group by61%and59%, respectively.4.5Body weight and Serum lipid profileThere was no significant difference in body weight among the five groups of ApoE-/-mice, which suggested lentiviral transfection was safe in our experimental animals. Similarly, serum levels of TC, TG, LDL-C and HDL-C did not change significantly in all the experimental groups.4.6The effect of NonO on carotid plaque disruptionH&E staining revealed that in carotid arteries of ApoE-/-mice, the plaque rupture rate was46.67%(7/15) in the control group, the si-N.C group and the N.C group respectively,13.33%(2/15) in the si-NonO group and66.67%(10/15) in the NonO group. Perl’s staining also verified the results by indicating intraplaque thrombi and hemorrhage.4.7NonO changed carotid plaque compositionWe detected whether NonO changed the compositions of carotid plaques including macrophages, lipid, SMCs and collagen. NonO knockdown significantly decreased the content of macrophages and lipid, but increased the content of SMCs and collagen in carotid plaques, whereas NonO overexpression exerted opposite effects. Accordingly, the plaque vulnerability index was significantly reduced by NonO knockdown but elevated by NonO overexpression. These results showed that NonO played a critical role in plaque destabilization.4.8Effect of NonO on the expression of P4Hal in vivoWestern blot showed that the protein level of P4Hal were significantly increased in the NonO-silencing mice, whereas they were remarkably decreased in the NonO-overexpression mice. The result confirmed that NonO participated in inhibiting the expression of P4Hα1.4.9Effect of NonO on the expression of MMP-2and MMP-9both in vivo and in vitroWe also detected the expression levels of MMP-2and MMP-9both in vivo and in vitro.In ApoE-/-mice and RAW264.7cells, the silencing of NonO downregulated the expression levels of MMP-2and MMP-9, while the overexpression of NonO upregulated the expression levels of them. In RAW264.7cells, we also detected the activity of MMP-2and MMP-9and found that NonO knockdown significantly suppressed the activity of MMP-2and MMP-9, whereas NonO overexpresion obviously increased them. 4.10Effect of NonO on proinflammatory cytokine expression both in vivo and in vitroIn ApoE-/-mice, the relative contents of IL-1β and IL-6in the carotid plaques analysed by immunohistochemistry were significantly lower in si-NonO group, whereas significantly higher in NonO-LV group than those in the control group. Western blot showed that NonO significantly increased the protein expression levels of LOX-1and COX-2, whereas NonO downregulation decreased their expression.In cultured RAW264.7cells, the protein expression levels of IL-1β, MCP-1, ICAM-1and VCAM-1were remarkably decreased in si-NonO group, whereas increased in NonO group compared with those in the control group. So, NonO promoted the expression of inflammatory cytokines both in vivo and in vitro.4.11NonO combined with NF-κB and influenced its nuclear translocation and phosphorylationCo-immunoprecipitation assays were performed in RAW264.7cells treated with or without TNF-a. Whole cell lysates were analyzed in SDS/PAGE along with anti-NF-κB p65or anti-NonO immunoprecipitations. Western blot showed that in the nonstimulated state, NonO coimmunoprecipitated with NF-κB p65, although the interaction was weak; after TNF-α stimulation, the interaction was significantly enhanced between NonO and NF-κB p65.We detected the effect of NonO on the nuclear translocation of NF-κB p65. Compared with the control group, si-NonO group showed significantly suppressed p65nuclear translocation, whereas NonO group remarkably increased this translocation. In addition, compared with the control group, NonO silencing could suppress the level of p-p65, while NonO overexpression could enhance it. We also detected the expression of IkBα and found that NonO overexpression significantly decreased the expression level of IκBα, whereas NonO silencing enhaced its protein level.5Conclusion(1) In ApoE-/-mice, the expression of NonO was upregulated in its atherosclerotic plaque. (2) NonO played an important role in plaque destabilization, and gene silencing of NonO could increase the stability of plaque.(3) NonO destabilizes atherosclerotic plaques via increasing the expression of MMP-2and MMP-9, suppressing the expression of P4Hα1and exaggerating inflammatory responses mediated by NF-κB. 1IntroductionDiabetic nephropathy (DN) has become the most common cause of end-stage renal disease and represents an increasing global public health problem. A wealth of evidence indicates that renin-angiotensin system (RAS) plays a key role in the pathogenesis of DN, and a galaxy of clinical trials have proven that angiotensin-converting enzyme (ACE) inhibitors and type1angiotensin receptor antagonists are effective in attenuating the development of DN. These protective effects were originally thought to result from blocking angiotensin Ⅱ-dependent pathways. However, the recent discovery of a homologue of ACE, ACE2, revealed a new pathway for angiotensin peptide metabolism. ACE2generates angiotensin(1-7)[Ang(1-7)] from angiotensin Ⅱ and plays a protective role against DN. However, the effect of Ang(1-7), the primary product of ACE2, on DN remains poorly understood.Ang(1-7) as a heptapeptide is mainly derived from the degradation of Angiotensin Ⅱ by ACE2in the kidney. Consistent with its site of synthesis, Ang(1-7) exerts important effects on renal homeostasis. In proximal tubular cells, Ang(1-7) activates tyrosine phosphatase and inhibits high-glucose-stimulated p38MAPK, thereby suppressing high-glucose-induced protein synthesis. Ang(1-7) attenuates renal vascular dysfunction by alleviating NADPH oxidase (NOX)-mediated oxidative stress. However, an recent study showed that an injection of moderate dose of Ang(1-7) paradoxically accelerated STZ-induced diabetic renal injury.So far, several important issues remain unsolved. First, whether Ang(1-7) treatment is beneficial or harmful to DN is controversial. Second, the effect of different doses of Ang(1-7) on DN is to be defined. Third, it is unclear whether combined treatment with Ang(1-7) and an angiotensin receptor blocker (ARB) is superior to either treatment alone for DN. Finally, t are only poorly understood.2Objectives(1) To assess the effect of Ang(1-7) and the combined treatment of Ang(1-7) and ARB on STZ-induced diabetic renal injury.(2) To elucidate the possible mechanisms underlying the effect of Ang(1-7) on DN.3Methods3.1Animal Model120male Wistar rats (10weeks old,200to250g) were randomly divided into two groups:control group (n=15) that received an intraperitoneal injection of normal saline and DM group (n=105) that received intraperitoneal injection of65mg/kg STZ. The status of DM in rats was confirmed by a tail-blood glucose level higher than16.7mmol/148h after STZ injection. All diabetic rats received an intraperitoneal injection of insulin (2-3U) every3days to maintain blood glucose levels between16.7and25mmol/1to prevent mouse death induced by excessively high blood glucose levels. Twelve weeks after STZ injection, diabetic rats were further randomly divided into seven subgroups (n=15per subgroup) for treatment:no treatment subgroup (NT group); large-dose, moderate-dose and small-dose Ang(1-7) subgroups that received a subcutaneous injection of800ng/kg-min,400ng/kg-min and200ng/kg-min of Ang(1-7), respectively, by an embedded mini-osmotic pump; valsartan subgroup given by intragastric administration at a dose of30mg/kg per day; large-dose Ang(1-7)+valsartan (30mg/kg per day) subgroup and large-dose Ang(1-7)+A779(both800ng/kg-min) subgroup. After treatment for4weeks, all rats underwent euthanasia.3.2General condition of rats At the end of our experiment, the blood glucose, systolic pressure (SBP), body weight (BW), kidney weight (KW),24h urine volume, creatinine levels of blood and urine were measured and KW/BW and creatinine clearance were calculated.3.3ELISAELISA was performed to test Ang(1-7) levels of serum and urine, the level of urinary albumin, and24-h urinary albumin were calculated.3.4Measurement of MDA content and SOD activityThe content of MDA and the activity of SOD in glomeruli were measured according to the specifications of kits.3.5Histopathological analysisTissue section were stained with PAS staining and glomeruli sclerosis index (GSI) were calculated. Immunohistochemistry were performed to detect the content of type IV collagen, TGF-(31, VEGF, PCNA and macrophages in glomeruli.3.6Cell culture and treatmentRat glomerular mesangial HBZY-1cells were pretreated with three doses of Ang(1-7)(50,100and200nmol/1) or10-6mol/1valsartan or200nmol/1Ang(1-7)+10-6mol/1valsartan or200nmol/1Ang(1-7)+200nmol/1A779for1h, and then treated with high glucose (25mmol/1) for24h. In addition, in the group treated by Ang(1-7)+A779, cells was pretreated with A779for30min before Ang(1-7) treatment. After stimulation, cells were harvested for further study.3.7Western blot analysisWestern blot was performed to detect the expression levels of NOX4, p47phox, PKCα, PKCpβ2, TGF-β1, p-Smad3in glomeruli and NOX4. p47phox、TGF-β1、 p-Smad3、VEGF和type IV collagen in HBZY-1cells. In addition, PKC proteins in the membranous and cytosolic fractions of HBZY-1cells were purified using membrane and cytosol protein extraction kit and western blot were performed to detect their expression levels in membrane and cytoplasm.3.8DHE staining, DCF staining and EdU proliferation assayDHE and DCF staining were performed to detect the level of ROS in HBZY-1cells. Proliferating HBZY-1cells were detected by use of an EdU labeling kit. 4Results4.1Serum and urine levels of Ang(1-7)The serum and urinary levels of Ang(1-7) in seven groups of DM rats were significantly lower than in the control group of rats before Ang(1-7) treatment. However, these levels were dose-dependently increased in the six treatment groups.4.2Blood pressure and blood glucose levelsAt the end of experiment, systolic blood pressure (SBP) was significantly lower in all seven groups of DM rats than in the control group of rats, but there was no significant difference in SBP among seven groups of DM rats. The blood glucose levels in all seven groups of DM rats were markedly higher than in the control group of rats, but did not differ among seven groups of DM rats.4.3Ang(1-7) dose-dependently ameliorated renal functionBody weight and creatinine clearance were significantly lower in seven groups of DM rats than those in the control group of rats. However, these parameters were dose-dependently increased in all treatment groups of DM rats except for the large-dose Ang(1-7)+A779group in which the body weight and creatinine clearance were similar with those in the no treatment group. The ratio of kidney weight to body weight,24-h urinary volume, plasma creatinine level and24-h urinary albumin excretion were all significantly higher in seven groups of DM rats than in the control group of rats. These values were decreased in all treatme更多还原